Isolation And Verification Of Strains Producing Cold-active Enzymes From The Intestinal Tracts Of Cold-water Fish And Study On Enzymatic Properties

Object：With the development of science and technology, the enzymatic preparations requirements ofindustrial production and environmental restoration have been increasing. In order to meet this demand,more and more bacteria producing cold-active enzymes have been screened and isolated from both theSouth and North Poles, ocean depths, glaciers and frozen soils and so on. However, there have been fewreports about enzyme-producing capabilities of bacteria in the gut of cold-water fish. Comparing withaforementioned low temperature environments, microenvironments of cold-water fish gut possess featuresof natural anaerobic, nutrient rich et al. Thus, screening and isolation of strains producing cold-activeenzymes from the intestinal tracts of cold-water fish is having more practical value. Therefore, we expect toobtain strains producing cold-active enzymes to solve the source of cold-active enzymes.Methods：Based on the unique geographical environment in Xinjiang, strains producing enzymes wereisolated from the gut of cold-water fish from Sayram Lake and Irtysh River basin of Altay by pure culture,and sequencing of their system were undertaken. The strains with larger hydrolyzation halo and loweroptimum growth temperature were selected as original strains and the second screening were conducted.The strains producing cold-active enzymes were screened and study on enzymatic properties.Results：①Based on colonial morphology, colour and cellular morphology,50strains producing enzymes wereisolated from the intestinal tracts of cold-water fish from Sayram Lake and Irtysh River basin of Altay,among which15strains were identified as yest.39strains isolated from the intestinal tracts of cold-waterfish that were collected from the Irtysh River basin of Altay, including Tinca tinca L.(12bacteria, nineyeasts), Lota lota L.(three bacteria, three yeasts), Perca fluviatilis L.(five bacteria), Carassius auratusgibelio Bloch (six bacteria) and Scardinius erythrophthalmus L.(one bacterum). The hosts of the rest of11strains were collected from Sayram Lake, including Salvelinus fontinalis Mitchill (seven bacteria, threeyeasts) and Coregonus peled Gmelin (one bacterum). The strains producing enzymes were not found in theintestinal tracts of Oncorhynchus mykiss Walbaum and Oncorhynchus mykiss var. Walbaum. This studyshows that the amount of culturable strains producing enzymes was less in the intestinal tracts ofcold-water fish from Sayram Lake and Irtysh River basin of Altay.②Based on the phenotypic characteristics and the comparison of partial16S rDNA gene sequences,35bacteria producing enzymes were classified into six genera, including Pseudomonas (14strains), Aerom-onas (three strains), Serratia (11strains), Stenotrophomonas (five strains), Massilia (one strain) and Flavo-bacterium (one strain).15yeasts all were member of Sporobolomyces. The comparison of partial16SrDNA/26S rDNAgene sequences shows that strains producing enzymes isolated from he intestinal tracts ofcold-water fish were homologous highly.③According to whether fluorescence and hydrolysis circles formed or not, enzyme production abilityof50strains were detected on the plates. The results indicated all bacteria could produce proteases. It isinteresting to note that combined hydrolytic activity was also detected in many strains. P-Y-4、P-J-1、P-D-9、P-D-7and P-D-4-15presented protease and lipase activity, and P-Q-3、A-Q-1、P-Q-1、A-R-1andA-Y-1presented protease and amylase activity. The strain producing cellulases and three enzymes fail to befound.15yeasts all presented protease and amylase activity. In terms of the ability to produce enzymes,20strains were able to produce amylases, and the D/d value was not above3.45. There were five strains ableto produce lipases, but the fluorescence was weak on the plates when irradiated with UV light at350nm.All strains were able to produce proteases, and the biggest D/d value reached6.90. The study reveals thatthe strains producing proteases were predominant in the intestinal tracts of cold-water fish.④Study on enzymatic properties of P-D-10extracellular proteases shows that, the extracellular proteases of strain P-D-10were able to hydrolyze casein at0-60℃, with10℃and40℃two optimumreaction temperature. The protease was susceptible to heat and retained only20.14%enzyme activity at70℃for10min. The enzyme present enzyme activity range from pH4.0to10.0, and the optimum reactionpH was8.5. With the exception of Tween80, the rest of metal ions, surfactants, reductants and chelant allinhibited enzyme activity of the extracellular proteases of strain P-D-10in different extent. Comaring withmetal ions, surfactants, reductants and chelant presented significant inhibiting effect. The relative enzymeactivity is only2%in reaction system with β-ME. The result shows that P-D-10extracellular proteaseswere susceptible to heat and possessed lower optimum reaction temperature, so P-D-10extracellularproteases belong to the category of cold-active enzymes.